The ability to clone gene sequences has permitted inquiries into the structure and function of nucleic acids, and has resulted in an ability to express highly desired proteins, such as hormones, enzymes, receptors, antibodies, etc., in diverse hosts.
The most commonly used methods for cloning a gene sequence involve the in vitro use of site-specific restriction endonucleases, and ligases. In brief, these methods rely upon the capacity of the “restriction endonucleases” to cleave double-stranded DNA in a manner that produces termini whose structure (i.e., 3′ overhang, 5′ overhang, or blunt end) and sequence are both well defined. Any such DNA molecule can then be joined to a suitably cleaved vector molecule (i.e., a nucleic acid molecule, typically double-stranded DNA, having specialized sequences which permit it to be replicated in a suitable host cell) through the action of a DNA ligase. The gene sequence may then be duplicated indefinitely by propagating the vector in a suitable host. Methods for performing such manipulations are well-known (see, for example, Perbal, B. A Practical Guide to Molecular Cloning, John Wiley & Sons, NY, (1984), pp. 208-216; Sambrook, J., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982); Old, R. W. et al., In: Principles of Gene Manipulation, 2nd Ed., University of California Press, Los Angeles, (1981), all herein incorporated by reference).
In some cases, a gene sequence of interest is so abundant in a source that it can be cloned directly without prior purification or enrichment. In most cases, however, the relative abundance of a desired target DNA molecule will require the use of ancillary screening techniques in order to identify the desired molecule and isolate it from other molecules of the source material.
A primary screening technique involves identifying the desired clone based upon its DNA sequence via hybridization with a complementary nucleic acid probe.
In situ filter hybridization methods are particularly well known (see, Sambrook, J., et al., In: Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). In such methods, bacteria are lysed on the surface of the membrane filter and then incubated in the presence of a detectably labeled nucleic acid molecule whose sequence is complementary to that of the desired sequence. If the lysate contains the desired sequence, hybridization occurs and thereby binds the labeled molecule to the adsorbent surface. The detection of the label on the adsorbent surface reveals that the bacteria sampled contained the desired cloned sequence.
Although these screening methods are useful and reliable, they require labor-intensive and time consuming steps such as filter preparation and multiple rounds of filter hybridization and colony platings/phage infections. Generally, these procedures will screen up to 106 colonies effectively, but may take weeks or months to yield the desired clone.
Other approaches have been developed to isolate recombinant molecules which have eliminated the tedious filter-handling procedure. These approaches employ conventional hybridization technology coupled with chromatography or magnetic particle technology. Rigas, B. et al., for example, reported a method for isolating one plasmid species from a mixture of two plasmid species. In the disclosed method, circular double-stranded plasmid DNA is hybridized to a RecA protein-coated biotinylated probe to form a stable triple-stranded complex, which is then selectively bound to an agarose-streptavidin column (Rigas, B. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83: 9591-9595 (1986)). The method thus permits the isolation of cloned double-stranded molecules without requiring any separation of the strands.
A DNA isolation method, termed “triplex affinity capture,” has been described in which a specific double-stranded genomic DNA is hybridized to a biotinylated homopyrimidine oligonucleotide probe to form a “triplex complex,” which can then be selectively bound to streptavidin-coated magnetic beads (Ito, T. et al., Nucleic Acids Res. 20: 3524 (1992); Ito, T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89: 495-498 (1992)). Takabatake, T. et al. have described a variation of this technique that employs a biotinylated purine-rich oligonucleotide probe to detect and recover the desired nucleic acid molecule (Takabatake, T. et al., Nucleic Acid Res. 20: 5853-5854 (1992)). A practical drawback with these particular approaches is that they are restricted to isolation of target DNA sequences containing homopurine-homopyrimidine tracts.
Fry, G. et al. discuss a method for sequencing isolated M13-LacZ phagemids (Fry, G. et al., BioTechniques 13:124-131 (1992)). In this method, a clone is selected and the phagemid DNA is permitted to hybridize to a biotinylated probe whose sequence is complementary to the phagemid's lacZ region. The biotinylated probe is attached to a streptavidin-coated paramagnetic bead. Since the DNA bound to the bead can be separated from unbound DNA, the method provides a means for separating the cloned sequence from the bacterial sequences that are inevitably present (Fry, G. et al., BioTechniques 13:124-131 (1992)).
Still another method of screening recombinant nucleic acid molecules is described by Kwok, P. Y. et al. This method, which is an extension of PCR-based screening procedures uses an ELISA-based oligonucleotide-ligation assay (OLA) to detect the PCR products that contain the target source (Kwok, P. Y. et al., Genomics 13: 935-941 (1992)). The OLA employs an “reporter probe” and a phosphorylated/biotinylated “anchor” probe, which is captured with immobilized streptavidin (Landegren, U. et al., Science 241:1077-1080 (1988)).
The isolation of target DNA from a complex population using a subtractive hybridization technique has also been described (Lamar, E. E. et al., Cell 37:171-177 (1984); Rubenstein, J. L. R. et al., Nucleic Acids Res. 18:4833-4842 (1990); Hedrik, S. M. et al., Science 308:149-153 (1984); Duguid, J. R. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5738-5742 (1988)). In such “subtractive hybridization” screening methods, the cDNA molecules created from a first population of cells is hybridized to cDNA or RNA of a second population of cells in order to “subtract out” those cDNA molecules that are complementary to nucleic acid molecules present in the second population and thus reflect nucleic acid molecules present in both populations.
The method is illustrated by Duguid, J. R. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 85:5738-5742 (1988)) who used subtractive hybridization to identify gene sequences that were expressed in brain tissue as a result of scrapie infection. A cDNA preparation made from uninfected cells was biotinylated and permitted to hybridize with cDNA made from infected cells. Sequences in common to both cDNA preparations hybridized to one another, and were removed from the sample through the use of a biotin-binding (avidin) resin.
Weiland, I. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 87:2720-2724 (1990)) reported an improved method of subtractive hybridization in which tester DNA was cleaved with a restriction endonuclease, and then permitted to hybridize to sheared driver DNA at high C0t values (“C0t” is the product of the initial concentration of DNA and the time of incubation). By cloning the double-stranded, PCR-amplified, unique DNA molecules into a plasmid vector, it was possible to obtain an enrichment in the relative proportion of target sequences recovered.
Rubenstein, J. L. R. et al. (Nucleic Acids Res. 18:4833-4842 (1990)) reported a further improvement in subtractive hybridization methods that employed single-stranded phagemid vectors to provide both the target and tester DNA. In the method, hybridized phagemid DNA-biotinylated driver strand complexes are separated from unhybridized DNA by the addition of streptavidin. Unhybridized single-stranded DNA was subsequently converted to the double-stranded form using Taq DNA polymerase and an oligonucleotide complementary to a common region found within the single-stranded DNA. The use of this method is, however, limited by the need to follow a rigorous single-stranded phagemid purification protocol in order to obtain a preparation virtually free of contaminant double-stranded DNA (Rubenstein, J. L. R. et al., Nucleic Acids Res. 18: 4833-4841 (1990)).
In sum, methods for isolating particular target nucleic acid molecules are restricted by the abundance of the DNA target sequence, and by time-consuming steps. Accordingly, a method that would expedite the isolation of desired target nucleic acid molecules and that could yield essentially pure target DNA would be highly desirable.